Vaccine for the prevention of breast cancer relapse

ABSTRACT

The invention features methods to induce and maintain a protective cytotoxic T-lymphocyte response to a peptide of the HER2/neu oncogene, E75, with the effect of inducing and maintaining protective or therapeutic immunity against breast cancer in a patient in clinical remission. The methods comprise administering to the patient an effective amount of a vaccine composition comprising a pharmaceutically acceptable carrier, an adjuvant such as recombinant human GM-CSF, and the E75 peptide at an optimized dose and schedule. The methods further comprise administering an annual or semi-annual booster vaccine dose due to declining E75-specific T cell immunity. The invention also features vaccine compositions for use in the methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application No. 60/941,524filed Jun. 1, 2007, the entire disclosure of which is incorporatedherein by reference.

FIELD

The invention relates generally to the field of preventive andtherapeutic vaccines. More specifically, the invention relates topeptide vaccines for the treatment of breast cancer and the preventionof relapse in patients in breast cancer remission.

BACKGROUND

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference herein, in its entirety and for all purposes.

Breast cancer (BCa) is the most common cancer diagnosis in women and thesecond-leading cause of cancer-related death among women (Ries L A G, etal. (eds). SEER Cancer Statistics Review, 1975-2003, National CancerInstitute, Bethesda, Md.). Major advances in breast cancer treatmentover the last 20 years have led to significant improvement in the rateof disease-free survival (DFS). For example, therapies utilizingantibodies reactive against tumor-related antigens have been used toblock specific cellular processes in order to slow disease progress orprevent disease recurrence. Despite the recent advances in breast cancertreatment, a significant number of patients will ultimately die fromrecurrent disease. Thus, there is a need for treatments that prevent orslow or prohibit the development of recurrent disease.

Vaccines are an attractive model for such treatments and preventions dueto their ease of administration, and because of their high rate ofsuccess observed for infectious diseases. The basic concept ofconstructing a cancer vaccine is straightforward in theory. Thedevelopment of effective cancer vaccines for solid tumors in practice,however, has met with limited success. For example, one group attemptingto administer a peptide vaccine directed against metastatic melanomaobserved an objective response rate of only 2.6% (Rosenberg S A et al.(2004) Nat. Med. 10:909-15).

There are many potential explanations for this low success rate (CampoliM et al. (2005) Cancer Treat. Res. 123:61-88). For example, even if anantigen is specifically associated with a particular type of tumor cell,the tumor cells can express only low levels of the antigen, or it can belocated in a cryptic site or otherwise shielded from immune detection.In addition, tumors often change their antigenic profile by sheddingantigens as they develop. Also contributing to the low success rate isthe fact that tumor cells can express very low levels of MHC proteinsand other co-stimulatory proteins necessary to generate an immuneresponse.

Additional problems facing attempts at vaccination against tumors arisein patients with advanced-stage cancers. Such patients tend to havelarger primary and metastatic tumors, and the cells on the interior ofthe tumor can not be accessible due to poor blood flow. This isconsistent with the observation that vaccine strategies have tended tobe more successful for the treatment of hematologic malignancies(Radford K J et al. (2005) Pathology 37:534-50; and, Molldrem J J (2006)Biol. Bone Marrow Transplant. 12:13-8). In addition, as tumors becomemetastatic, they can develop the ability to release immunosuppressivefactors into their microenvironment (Campoli, 2005; and, Kortylewski Met al. (2005) Nature Med. 11:1314-21). Metastatic tumors have also beenassociated with a decrease in the number of peripheral bloodlymphocytes, and dendritic cell dysfunction (Gillanders W E et al.(2006) Breast Diseases: A Year Book and Quarterly 17:26-8).

While some or all of these factors can contribute to the difficulty indeveloping an effective preventative or therapeutic vaccine, the majorunderlying challenge is that most tumor antigens are self antigens orhave a high degree of homology with self antigens, and are thus expectedto be subject to stringent immune tolerance. Thus, it is clear that manypeptide-based cancer vaccines, with or without immune-stimulatingadjuncts, can be doomed to only limited success in clinical practice dueto low immunogenicity and lack of specificity.

Prototype breast cancer vaccines based on single antigens have beenmoderately successful in inducing a measurable immune response in animalexperiments and in clinical tests with breast cancer patients. Theobserved immune response, however, has not translated into aclinically-significant protective immunity against resurgence of diseaseput in remission by standard surgery and chemotherapy. Thus, novelvaccine approaches are needed to further improve recurrence rates andoverall survival among BCa patients.

Preferred vaccine epitopes are those that are expressed exclusively, orat least at increased levels by a neoplasm. HER2/neu is a proto-oncogeneexpressed in many epithelial malignancies (Slamon D J et al. (1989)Science 244:707-12). Gene amplification and overexpression of theHER2/neu protein is found in 20-25% of BCa, and its excess presence isan indicator of poor prognosis (Pritchard K I et al. (2006) N. Engl. J.Med. 354:2103-11). HER2/neu has been studied fairly extensively, andseveral immunogenic peptides have been identified from this protein. Onesuch peptide is termed E75, and corresponds to amino acids 369-377 ofHER2/neu (SEQ ID NO:1) (U.S. Pat. No. 6,514,942).

Attempts have been made to utilize E75 as an anti-cancer vaccine, forexample, as a single peptide vaccine combined with differentimmunoadjuvants (Zaks T Z et al. (1998) Cancer Res. 58:4902-8; Knutson KL et al. (2002) Clin. Cancer Res. 8:1014-8; and, Murray J L et al.(2002) Clin. Cancer Res. 8:3407-18); loaded on to autologous dendriticcells and reinfused (Brossart P et al. (2000) Blood 96:3102-8; and, KonoK et al. (2002) Clin. Cancer Res. 8:3394-3400); or embedded in longerpeptides capable of binding HLA class II molecules in order to recruitCD4 helper T-cells (Disis M L et al. (1999) Clin. Cancer Res. 5:1289-97;and, Disis M L et al. (2002) J. Clin. Oncol. 20:2624-32). Each approachhas stimulated an E75-specific cytotoxic T cell-mediated immuneresponse, but has not demonstrated a clinically significant therapeuticor protective immunity in women with advanced stage breast cancer.

HER2/neu is a member of the epidermal growth factor receptor family andencodes a 185-kd tyrosine kinase receptor involved in regulating cellgrowth and proliferation. (Popescu N C, King C R, Kraus M H.Localization of the human erbB-2 gene on normal and rearrangedchromosome 17 to bands q12-21.32. Genomics 1989; 4:362-366; Yarden Y,Sliwkowski M X. Untangling the ErbB signaling network. Nat Rev Mol CellBio 2001; 2:127-137.) Over-expression and/or amplification of HER2/neuis found in 25-30% of invasive breast cancers (BCa) and is associatedwith more aggressive tumors and a poorer clinical outcome. (Slamon D J,Clark G M, Wong S G, et al. Human breast cancer: correlation of relapseand survival with amplification of the HER-2/neu oncogene. Science 1987;235:177-182; Slamon D J, Godolphin W, Jones L A, et al. Studies of theHER-2/neu proto-oncogene in human breast and ovarian cancer. Science1989; 244:707-12; Toikkanen S, Helin H, Isola J, Joensuu H. Prognosticsignificance of HER-2 oncoprotein expression in breast cancer: A 30-yearfollow-up. J Clin Oncol 1992; 10:1044-1048.)

Determining HER2/neu status is performed predominately via two tests,immunohistochemistry (IHC) and fluorescence in situ hybridization(FISH). IHC detects over-expression of HER2/neu protein and is reportedon a semi-quantitative scale of 0 to 3+ (0=negative, 1⁺=low expression,2⁺=intermediate, and 3⁺=over-expression). FISH on the other hand detectsamplification (excess copies) of the HER2/neu gene and is expressed as aratio of HER2/neu gene copies to chromosome 17 gene copies andinterpreted as “over-expression” if FISH is ≧2.0 copies. (Hicks D G,Tubbs R R. Assessment of the HER2 status in breast cancer byfluorescence in situ hybridization: a technical review with interpretiveguidelines. Hum Pathol 2005; 36:250-261.) Concurrence rate of IHC andFISH is approximately 90%. (Jacobs T W, Gown A M, Yaziji H, et al.Specificity of HercepTest in determining HER-2/neu status of breastcancers using the United States Food and Drug Administration-approvedscoring system. J Clin Oncol 1999; 17:1533-1541.) FISH is considered thegold standard, as retrospective analysis reveals it is a betterpredictor of trastuzumab (Tz) response; it is more objective andreproducible. (Press M F, Slamon D J, Flom K J, et al. Evaluation ofHER-2/neu Gene Amplification and Overexpression: Comparison ofFrequently Used Assay Methods in a Molecularly Characterized Cohort ofBreast Cancer Specimens. J Clin Oncol 2002; 14:3095-3105; Bartlett J,Mallon E, Cooke T. The clinical evaluation of HER-2 status: which testto use? J Pathol 2003; 199:411-417; Wolff A C, Hammond M E H, Schwartz JN, et al. American Society of Clinical Oncology/College of AmericanPathologists guideline recommendations for human epidermal growth factorreceptor 2 testing in breast cancer. J Clin Oncol 2007; 25:118-145.)

Identification and quantification of HER2/neu as a proto-oncogene hasled to humoral or antibody-based passive immunotherapy, to include theuse of Tz (Herceptin®). Tz is a recombinant, humanized monoclonalantibody that binds the extracellular juxtamembrane domain of HER2/neuprotein. (Plosker G L, Keam S J. Trastuzumab: A review of its use in themanagement of HER2-positive metastatic and early-stage breast cancer.Drugs 2006; 66:449-475.) Tz is indicated for HER2/neu over-expressing(IHC 3 or FISH ≧2.0) node-positive (NP) and metastatic BCa patients,(Vogel C L, Cobleigh M A, Tripathy D, et al. Efficacy and safety oftrastuzumab as a single agent in first-line treatment ofHER2-overexpressing metastatic breast cancer. J Clin Oncol 2002;20:719-726; Piccart-Gebhart M J, Procter M, Leyland-Jones B, et al.Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer.N Engl J Med 2005; 353:1659-1672) and shows very limited activity inpatients with low to intermediate HER2/neu expression. (Herceptin(Trastuzumab) prescription product insert. South San Francisco, Calif.:Genentech Inc; revised September 2000.)

Another form of immunotherapy being pursued is vaccination and activeimmunotherapy targeting a cellular immune response to epitopes on tumorassociated antigens (TAA) such as HER2/neu. HER2/neu is a source ofseveral immunogenic peptides that can stimulate the immune system torecognize and kill HER2/neu-expressing cancer cells. (Fisk B, Blevins TL, Wharton J T, et al. Identification of immunodominant peptide of theHER2/neu proto-oncogene recognized by ovarian tumor-specific CTL lines.J Exp Med 1995; 181:2109-2117.)

E75 (KIFGSLAFL, HER2/neu, 369-377) is a peptide sequence in the HER2/neuproto-oncogene family and is in use in clinical trials as an anti-cancervaccine to stimulate cytotoxic T lymphocytes (CTL) to destroy cancercells. (Zaks, T. et. al. Immunization with a peptide epitope (369-377)from HER-2/neu leads to peptide specific cytotoxic T lymphocytes thatfail to recognize HER-2/neu+ tumors. Cancer Research. 58 (21): 4902-8.1998; Knutson K L, Schiffman K, Cheever M A, et al: Immunization ofcancer patients with HER-2/neu, HLA-A2 peptide, p 369-377, results inshort-lived peptide-specific immunity. Clin Cancer Res 8:1014-1018,2002; Murray J L, Gillogly M E, Przepiorka D, et al: Toxicity,immunogenicity, and induction of E75-specific tumorlytic CTLs by HER-2peptide E74 (369-377) combined with granulocyte macrophagecolony-stimulating factor in HLA-A2+ patients with metastatic breast andovarian cancer. Clin Cancer Res 8:3407-3418, 2002; Avigan D, Vasir B,Gong J, et al. Fusion cell vaccination of patients with metastaticbreast and renal cell cancer induces immunological responses. ClinCancer Res 2004: 10:4699-4708; Disis M L, Gooley T A, Rinn K, et al.Generation of T-cell immunity to the HER2/neu protein after activeimmunization with HER2/neu peptide-based vaccines. J Clin Oncol 2002;20:2624-32; Disis M L, Grabstein K H, Sleath P R, et al. Generation ofimmunity to the HER-2/neu oncogenic protein in patients with breast andovarian cancer using a peptide-based vaccine. Clin Cancer Res5:1289-1297, 1999.

Targeted passive immunotherapy based on the HER2/neu proto-oncogene hasprimarily revolved around the use of Tz (Herceptin®). Tz is arecombinant, humanized monoclonal antibody that binds the extracellularjuxtamembrane domain of the HER2/neu protein. Tz is approved byregulatory authorities and indicated for treatment of HER2/neuover-expressing (IHC 3+ or FISH >2.0) tumors in metastatic breast cancerpatients and in the adjuvant setting for node-positive breast cancerpatients. Tz has undergone multiple clinical trials and is now routinelyused in the treatment of metastatic patients and in the adjuvanttreatment of high risk breast cancer patients with overexpression ofHER2/neu. Tz, however, shows limited activity in patients with low tointermediate HER2/neu expression. Therefore, based on the previousresults seen with Tz, immunogenic peptide vaccines targeting HER2/neuwould not be expected to be effective in cancer patients with low andintermediate levels of HER2/neu tumor expression.

Thus, there is a need in the art to exploit the immunoprotective andtherapeutic potential of E75 to produce vaccines that offer breastcancer patients in clinical remission reliable protection againstrecurrence of the disease.

SUMMARY

The invention features methods of inducing and maintaining immunityagainst breast cancer relapse in patients in breast cancer clinicalremission. The methods comprise administering to the patient aneffective amount of a composition comprising a pharmaceuticallyeffective carrier and a peptide having the amino acid sequence SEQ IDNO:2. The administration can be accomplished by any means suitable inthe art, such as inoculation or injection, and more particularlyintradermal injection, which can occur with one or more separate doses.Such doses can comprise an equal concentration of the peptide and animmunoadjuvant, can be administered substantially concurrently, and canbe administered at one inoculation site or spaced apart from each otheron the surface of the skin. The composition can be administeredapproximately three to six times or more on a monthly basis until theprotective immunity is established. In some aspects, the compositionfurther comprises an adjuvant such as recombinant human granulocytemacrophage-colony stimulating factor (GM-CSF).

In some aspects, the methods further comprise administering to thesubject a booster vaccine dose, which comprises an effective amount of acomposition comprising a pharmaceutically effective carrier and apeptide having SEQ ID NO:2. In some aspects, the composition of thebooster further comprises an adjuvant such as GM-CSF. The administrationof a booster can be carried out by inoculation or injection, and can becan be administered every six or 12 months thereafter.

The patient can be any mammal, and is preferably a human. In certainaspects, the human is positive for major histocompatibility antigenblood-typed as human leukocyte antigen A2 or human leukocyte antigen A3.In other aspects, the human is positive for the expression of detectablelevels of HER2/neu. In some aspects, the human is a low or intermediateHER2/neu-expressor. For example, in some preferred aspects, the humanhas a immunohistochemistry (IHC) rating of 1+ or 2+ and/or afluorescence in situ hybridization (FISH) rating of less than 2.0. Inother aspects, the human can have an IHC rating up to 3+. In otheraspects, the human can be overexpressors of HER2/neu. For example, insome preferred aspects, the human has an immunohistochemistry (IHC)rating of 3+ and/or a fluorescence in situ hybridization (FISH) ratingof greater than or equal to 2.0.

The invention also features compositions for use in the inventivemethods. Such compositions comprise a pharmaceutically acceptablecarrier, an effective amount of a peptide having the amino acid sequenceSEQ ID NO:2, an adjuvant such as granulocyte macrophage-colonystimulating factor, and an optimized immunization schedule. In somespecific aspects, the preferred concentrations and schedules of thevaccine composition include: (1) 1 mg/ml peptide and 0.25 mg/mladjuvant, (2) 0.5 mg/ml peptide and 0.25 mg/ml adjuvant, (3) 0.1 mg/mlpeptide and 0.25 mg/ml adjuvant, and (4) 0.5 mg/ml peptide and 0.125mg/ml adjuvant, each with monthly inoculations for 6 consecutive monthsfollowed by annual booster inoculations for 3 or more years.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are included to provide a furtherunderstanding of the invention and are incorporated in and constitute apart of this specification, illustrate aspects of the invention andtogether with the description serve to explain the principles of theinvention. In the drawings:

FIG. 1 shows the maximum local and systemic toxicity experienced bypatients vaccinated with E75. Local toxicity (erythema and induration atinjection site) is a desired effect showing a response to the vaccine.The most common grade 2 local toxicities were pruritis or discomfortrequiring medication. Most common systemic toxicities were bone pain,flu-like symptoms and fatigue (commonly associated with GM-CSF) andlasted <24 hours. The two grade 3 systemic toxicities were angioedema ofthe tongue (after sixth inoculation) and bony pain.

FIG. 2 shows Kaplan Meier disease-free survival curves at 20 monthsmedian follow-up. For 171 enrolled patients, the recurrence rate in thevaccinated group was 5.6% compared to 14.2% in the observation group(P=0.04) at a median follow-up of 20 months. The disease-free survivalrates in the vaccinated and control groups were 92.5% and 77%,respectively.

FIGS. 3A and 3B show the vaccine-induced E75 CTL response. (A)Vaccine-induced E75-specific CTL for all patients. The median levels ofCD8+E75-specific CTL were significantly increased from pre-vaccinationlevels (0.39%, range 0-3.28%) to a maximum level (1.8, range 0.4-12.2%,P<0.0001), and post-vaccination level (0.70%, range 0.06-2.91%,P=0.002). There was no difference between pre-vaccine levels andlong-term (six month) levels of specific CD8+ T-cells. (B)Vaccine-induced E75-specific CTL based on pre-existing immunity.Patients with and without pre-existing immunity showed identicalpatterns in response to E75 vaccination with similar median maximum andpost-vaccination levels achieved for both. However, in those patientswithout pre-existing immunity, there was a significant increase in dimerlevels from pre-vaccine to six months post-vaccine (0.13% [range0-0.28%] vs. 0.45% [0-2.68%], P<0.0001).

FIGS. 4A to 4D show the results of delayed type hypersensitivity test.(A) DTH for all patients post-vaccination. Control 2.1±0.5 mm comparedto peptide 14.0±1.4 mm, P<0.0001. (B) Pre- and post-vaccine DTH for NNpatients. There was no difference in saline control vs. peptidepre-vaccination. Post-vaccination, there was a significant increase inDTH response to E75 peptide as compared to post-vaccine control(P<0.001) and compared to pre-vaccination E75 DTH (P<0.001). (C)Post-vaccination DTH by trial. NP patients had significantly larger DTHresponses as compared to NN patients (17.3±2.4 mm vs. 10.9±1.5 mm,P=0.02). This can be due to a difference in the median total vaccinedose in the NN group (2000 μg vs. 4000 μg, P<0.0001). (D)Post-vaccination DTH by dose groups. Patients receiving <6000 μg E75 hadsignificantly smaller DTH responses compared to patients receiving atotal of 6000 μg. (13.3±1.9 mm vs. 25.1±4.0 mm, P=0.008).

FIG. 5 shows the levels of CD8⁺ T-cells in booster patients. Patientsreceiving a booster 6 months after primary vaccination series hadsignificantly higher levels of CD8⁺ T-cells than patients >6 months fromprimary vaccination series. Among those patients >6 months, theydemonstrated a non-significant decline from 0.7% to 0.44% from their ownlevels at 6 months post-primary vaccination.

FIG. 6 shows graded local and systemic toxicity. The majority ofpatients experienced grade 1 local toxicity with only 2 patientsexperiencing grade 2 local toxicity. Over half of the patients had nosystemic toxicity and there were no grade 2 or 3 systemic toxicities.The eleven patients who experienced grade 1 systemic toxicity included(number of instances): fatigue (4), headache (4), myalgias (3), chills(2), fever (2), diarrhea (1), malaise (1), bone pain (1), andarthralgias (1).

FIG. 7 shows the booster response in patients lacking SRI showed a trendtowards an increasing number of antigen specific CD8⁺ T-cells.

FIG. 8 shows patients demonstrating increased IFN-γ secreting cellsdetected by enzyme-linked immunoabsorbance. Overall, 91% of patientsshowed increased antigen-specific (functional) T-cells as measured byELISPOT, with 50% showing a definite increase (increased IFN-γ secretingcells on ≧50% of assays).

FIG. 9 shows local reactions in booster patients. Patients receiving thebooster temporally closer to finishing their primary vaccination series(≦9 months; light bars) had significantly larger LR than thosepatients >9 months from their primary vaccination series. The two groupshad similar LR at the end of the primary series (left side). These datasuggest an additive effect of the booster in patients receiving boostersooner and a maintenance effect for patients receiving the boosterlater.

FIG. 10 shows the increasing LR over course of primary series, andillustrates that the two groups were the same in initial series and thatthe only difference is time from primary series. The vax 6 number isdifferent from Last prior LR number shown in FIG. 7 because somepatients only received 4 inoculations. The two groups were statisticallyidentical at all points except vaccine 3 when the ≦9 month group waslarger (97 vs. 80, p=0.04).

FIG. 11A to FIG. 11D show immunologic (mean±SE) and clinical responses(absolute recurrence and mortality rates) of patients enrolled in E75Phase II trial by HER2/neu LE vs. OE.

A. In vitro immune response—all in vitro pre-max % specific CD8+ T-cellsstatistically increased (LE p<0.001, OE p<0.001) and LE patients hadincreased max response compared to OE patients (p=0.04).

B. In vivo immune response—all in vivo pre-post DTHs statisticallyincreased (LE p<0.001, OE p=0.02).

C. Recurrence rates—recurrence rates were decreased in vaccinated LE andOE patients, albeit not statistically significant.

D. Mortality rates—vaccinated LE patients had a trend towards decreasedmortality rates (p=0.08).

FIG. 12A to FIG. 12D show immunologic (mean±SE) and clinical responses(absolute recurrence and mortality rates) of patients enrolled in E75Phase II trial by HER2/neu IHC expression level (0, 1+, 2+, 3+).

A. In vitro immune response—all in vitro pre-max % specific CD8+ T-cellsstatistically increased, whereas only HER2/neu 1+ pre-long term trendedtowards significance (p=0.08).

B. In vivo immune response—all in vivo pre-post DTHs statisticallyincreased (0 p=0.03. 1+ p=0.02, 2+ p=0.02, 3+ p=0.05).

C. Recurrence rates—recurrence rates were decreased in all vaccinatedIHC levels, albeit not statistically significant.

D. Mortality rates—mortality rates decreased in all vaccinated IHClevels and was statistically significant in HER2/neu IHC 1+ vaccinepatients (p=0.04).

FIGS. 13A and 13B show Dimer Assay and DTH per ODG vs. SDG. (A) Asignificant difference in the ODG vs. SDG was seen in the averagepre-vaccine CD8+E75-specific T cells levels (0.91+0.13% vs. 0.54+0.11%,p=0.03). No significant difference seen between the average maximumCD8+E75-specific T cell levels. The optimal dose showed a trend towardan increase in the average of monthly post vaccination percent ofCD8+E75-specific T cells (0.87+0.10% vs. 0.67+0.05%, p=0.07). Nodifference seen in the average long term CD8+E75-specific T cell levelsbetween groups at 6 months. (B) Orthogonal mean DTH response (mm)between the ODG vs. SDG showed no difference to the control inoculum(3.0+1.1 mm vs. 2.0+0.5 mm). DTH response to the peptide wassignificantly elevated in the ODG vs. the SDG (21.5+2.5 mm vs. 11.3+1.3mm, p=0.00021).

FIG. 14 shows comparison of the clinical recurrence rates between theSDG and ODG. Compared to the SDG, the ODG demonstrated a trend towardlower recurrence rates (p=0.27) but at a significantly shorter medianfollow-up. However, the ODG consisted of younger patients withsignificantly more aggressive disease.

DETAILED DESCRIPTION

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Such termsare to be given their ordinary meaning in the art unless otherwiseindicated. Other specifically defined terms are to be construed in amanner consistent with the definition provided herein.

The term “prevent” refers to any success or indicia of success in theforestalling of breast cancer recurrence/relapse in patients in clinicalremission, as measured by any objective or subjective parameter,including the results of a radiological or physical examination.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound, material,or composition, as described herein effective to achieve a particularbiological result such as, but not limited to, biological resultsdisclosed, described, or exemplified herein. Such results can include,but are not limited to, the prevention of breast cancer, and moreparticularly, the prevention of recurrent breast cancer, e.g., theprevention of relapse in a subject, as determined by any means suitablein the art. Optimal therapeutic amount refers to the dose, schedule andthe use of boosters to achieve the best therapeutic outcome.

“Pharmaceutically acceptable” refers to those properties and/orsubstances which are acceptable to the patient from apharmacological/toxicological point of view and to the manufacturingpharmaceutical chemist from a physical/chemical point of view regardingcomposition, formulation, stability, patient acceptance andbioavailability. “Pharmaceutically acceptable carrier” refers to amedium that does not interfere with the effectiveness of the biologicalactivity of the active ingredient(s) and is not toxic to the host towhich it is administered.

“Protective immunity” or “protective immune response,” means that thesubject mounts an active immune response to an immunogenic component ofan antigen such as the breast cancer antigens described and exemplifiedherein, such that upon subsequent exposure to the antigen, the subject'simmune system is able to target and destroy cells expressing theantigen, thereby decreasing the incidence of morbidity and mortalityfrom recurrence of cancer in the subject. Protective immunity in thecontext of the present invention is preferably, but not exclusively,conferred by T lymphocytes.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

“Peptide” refers to any peptide comprising two or more amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres. Polypeptide refers to both short chains, commonlyreferred to as peptides, oligopeptides or oligomers, and to longerchains, generally referred to as proteins. Polypeptides can containamino acids other than the 20 gene-encoded amino acids. Polypeptidesinclude amino acid sequences modified either by natural processes, suchas post-translational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in apolypeptide, including the peptide backbone, the amino acid side-chainsand the amino or carboxyl termini. It will be appreciated that the sametype of modification can be present in the same or varying degrees atseveral sites in a given polypeptide. Also, a given polypeptide cancontain many types of modifications. Polypeptides can be branched as aresult of ubiquitination, and they can be cyclic, with or withoutbranching. Cyclic, branched and branched cyclic polypeptides can resultfrom natural posttranslational processes or can be made by syntheticmethods. Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination.

“Booster” refers to a dose of an immunogen administered to a patient toenhance, prolong, or maintain protective immunity and to overcome thedown-regulation of T-cell responses mediated by regulatory T-cells.

“Free of breast cancer” or “disease free” or NED (No Evidence ofDisease) means that the patient is in clinical remission induced bytreatment with the current standard of care therapies. By “remission” or“clinical remission,” which are used synonymously, it is meant that theclinical signs, radiological signs, and symptoms of breast cancer havebeen significantly diminished or have disappeared entirely based onclinical diagnostics, although cancerous cells can still exist in thebody. Thus, it is contemplated that remission encompasses partial andcomplete remission. The presence of residual cancer cells can beenumerated by assays such as CTC (Circulating Tumor Cells) and can bepredictive of recurrence.

“Relapse” or “recurrence” or “resurgence” are used interchangeablyherein, and refer to the radiographic diagnosis of return, or signs andsymptoms of return of breast cancer after a period of improvement orremission.

Breast cancer is a major health concern for women worldwide. Breastcancer vaccines that have been attempted to date have been limited inefficacy, particularly with respect to preventing relapse indisease-free patients. In accordance with the present invention, it hasbeen determined that recurrence of breast cancer in disease-freepatients can be effectively prevented by administration to the patientof a peptide of the HER2/neu oncogene, E75 (SEQ ID NO:2) under certainconditions. It has also been unexpectedly determined that the E75peptide is associated with MHC HLA-A2 and -A3, and thus can induceprotective immunity in patients having the HLA-A2 and -A3 haplotype.

Accordingly, the present invention features vaccine compositions forinducing protective immunity against breast cancer relapse. Theinvention also features methods for inducing and for maintainingprotective immunity against breast cancer, and more particularly againstrecurrent breast cancer. In some aspects, the methods compriseadministering to a subject an effective amount of a compositioncomprising a pharmaceutically effective carrier and a polypeptide havingthe amino acid sequence SEQ ID NO:2. Variants of SEQ ID NO:2, includingthose with modified side chains of amino acids as described by U.S. Pat.Publ. No. 20050169934 are suitable for use as an immunogen in theinventive vaccine compositions and methods.

The subject can be any animal, and preferably is a mammal such as ahuman, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow,horse, pig, and the like. Humans are most preferred. In highly preferredaspects, the humans are positive for the HLA-A2 or HLA-A3 haplotypes. Inother preferred aspects, the humans are positive for the expression ofhuman HER2/neu, including preferentially humans with low and/orintermediate HER2/neu expressing tumors, as well as humans that areoverexpressors of HER2/neu.

The vaccine compositions can be formulated as freeze-dried or liquidpreparations according to any means suitable in the art. Non-limitingexamples of liquid form preparations include solutions, suspensions,syrups, slurries, and emulsions. Suitable liquid carriers include anysuitable organic or inorganic solvent, for example, water, alcohol,saline solution, buffered saline solution, physiological salinesolution, dextrose solution, water propylene glycol solutions, and thelike, preferably in sterile form.

The vaccine compositions can be formulated in either neutral or saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the active polypeptides) and whichare formed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or organic acids such as acetic, oxalic, tartaric,mandelic, and the like. Salts formed from free carboxyl groups can alsobe derived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine, and the like.

The vaccine compositions are preferably formulated for inoculation orinjection into the subject. For injection, the vaccine compositions ofthe invention can be formulated in aqueous solutions such as water oralcohol, or in physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. Thesolution can contain formulatory agents such as suspending, preserving,stabilizing and/or dispersing agents. Injection formulations can also beprepared as solid form preparations which are intended to be converted,shortly before use, to liquid form preparations suitable for injection,for example, by constitution with a suitable vehicle, such as sterilewater, saline solution, or alcohol, before use.

The vaccine compositions can also be formulated in sustained releasevehicles or depot preparations. Such long acting formulations can beadministered by inoculation or implantation (for example subcutaneouslyor intramuscularly) or by injection. Thus, for example, the vaccinecompositions can be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Liposomes and emulsions are well-known examplesof delivery vehicles suitable for use as carriers.

The vaccine compositions can comprise agents that enhance the protectiveefficacy of the vaccine, such as adjuvants. Adjuvants include anycompound or compounds that act to increase a protective immune responseto the E75 peptide antigen, thereby reducing the quantity of antigennecessary in the vaccine, and/or the frequency of administrationnecessary to generate a protective immune response. Adjuvants caninclude for example, emulsifiers, muramyl dipeptides, pyridine, aqueousadjuvants such as aluminum hydroxide, chitosan-based adjuvants, and anyof the various saponins, oils, and other substances known in the art,such as Amphigen, LPS, bacterial cell wall extracts, bacterial DNA, CpGsequences, synthetic oligonucleotides and combinations thereof (Schijnset al. (2000) CUM Opin. Immunol. 12:456), Mycobacterialphlei (M. phlei)cell wall extract (MCWE) (U.S. Pat. No. 4,744,984), M. phlei DNA(M-DNA), and M-DNA-M. phlei cell wall complex (MCC). Compounds which canserve as emulsifiers include natural and synthetic emulsifying agents,as well as anionic, cationic and nonionic compounds. Among the syntheticcompounds, anionic emulsifying agents include, for example, thepotassium, sodium and ammonium salts of lauric and oleic acid, thecalcium, magnesium and aluminum salts of fatty acids, and organicsulfonates such as sodium lauryl sulfate. Synthetic cationic agentsinclude, for example, cetyltrhethylammonlum bromide, while syntheticnonionic agents are exemplified by glycerylesters (e.g., glycerylmonostearate), polyoxyethylene glycol esters and ethers, and thesorbitan fatty acid esters (e.g., sorbitan monopalmitate) and theirpolyoxyethylene derivatives (e.g., polyoxyethylene sorbitanmonopalmitate). Natural emulsifying agents include acacia, gelatin,lecithin and cholesterol.

Other suitable adjuvants can be formed with an oil component, such as asingle oil, a mixture of oils, a water-in-oil emulsion, or anoil-in-water emulsion. The oil can be a mineral oil, a vegetable oil, oran animal oil. Mineral oils are liquid hydrocarbons obtained frompetrolatum via a distillation technique, and are also referred to in theart as liquid paraffin, liquid petrolatum, or white mineral oil.Suitable animal oils include, for example, cod liver oil, halibut oil,menhaden oil, orange roughy oil and shark liver oil, all of which areavailable commercially. Suitable vegetable oils, include, for example,canola oil, almond oil, cottonseed oil, corn oil, olive oil, peanut oil,safflower oil, sesame oil, soybean oil, and the like. Freund's CompleteAdjuvant (FCA) and Freund's Incomplete Adjuvant (FIA) are two commonadjuvants that are commonly used in vaccine preparations, and are alsosuitable for use in the present invention. Both FCA and FIA arewater-in-mineral oil emuslsions; however, FCA also contains a killedMycobacterium sp.

Immunomodulatory cytokines can also be used in the vaccine compositionsto enhance vaccine efficacy, for example, as an adjuvant. Non-limitingexamples of such cytokines include interferon alpha (IFN-α),interleukin-2 (IL-2), and granulocyte macrophage-colony stimulatingfactor (GM-CSF), or combinations thereof. GM-CSF is highly preferred.

Vaccine compositions comprising E75 peptide antigens and furthercomprising adjuvants can be prepared using techniques well known tothose skilled in the art including, but not limited to, mixing,sonication and microfluidation. The adjuvant can comprise from about 10%to about 50% (v/v) of the vaccine composition, more preferably about 20%to about 40% (v/v), and more preferably about 20% to about 30% (v/v), orany integer within these ranges. About 25% (v/v) is highly preferred.

Administration of the vaccine compositions can be by infusion orinjection (e.g., intravenously, intramuscularly, intracutaneously,subcutaneously, intrathecal, intraduodenally, intraperitoneally, and thelike). The vaccine compositions can also be administered intranasally,vaginally, rectally, orally, or transdermally. Additionally, vaccinecompositions can be administered by “needle-free” delivery systems.Preferably, the compositions are administered by intradermal injection.Administration can be at the direction of a physician or physicianassistant.

The injections can be split into multiple injections, with such splitinoculations administered preferably substantially concurrently. Whenadministered as a split inoculation, the dose of the immunogen ispreferably, but not necessarily, proportioned equally in each separateinjection. If an adjuvant is present in the vaccine composition, thedose of the adjuvant is preferably, but not necessarily, proportionedequally in each separate injection. The separate injections for thesplit inoculation are preferably administered substantially proximal toeach other on the patient's body. In some preferred aspects, theinjections are administered at least about 1 cm apart from each other onthe body. In some preferred aspects, the injections are administered atleast about 2.5 cm apart from each other on the body. In highlypreferred aspects, the injections are administered at least about 5 cmapart from each other on the body. In some aspects, the injections areadministered at least about 10 cm apart from each other on the body. Insome aspects, the injections are administered more than 10 cm apart fromeach other on the body, for example, at least about 12.5. 15, 17.5, 20,or more cm apart from each other on the body. Primary immunizationinjections and booster injections can be administered as a splitinoculation as described and exemplified herein.

Various alternative pharmaceutical delivery systems can be employed.Non-limiting examples of such systems include liposomes and emulsions.Certain organic solvents such as dimethylsulfoxide also can be employed.Additionally, the vaccine compositions can be delivered using asustained-release system, such as semipermeable matrices of solidpolymers containing the therapeutic agent. The various sustained-releasematerials available are well known by those skilled in the art.Sustained-release capsules can, depending on their chemical nature,release the vaccine compositions over a range of several days to severalweeks to several months.

To prevent breast cancer recurrence in a patient who is in breast cancerremission, a therapeutically effective amount of the vaccine compositionis administered to the subject. A therapeutically effective amount willprovide a clinically significant increase in the number of E75-specificcytotoxic T-lymphocytes (CD8⁺) in the patient, as well as a clinicallysignificant increase in the cytotoxic T-lymphocyte response to theantigen, as measured by any means suitable in the art. In the patient onthe whole, a therapeutically effective amount of the vaccine compositionwill destroy residual microscopic disease and significantly reduce oreliminate the risk of recurrence of breast cancer in the patient.

The effective amount of the vaccine composition can be dependent on anynumber of variables, including without limitation, the species, breed,size, height, weight, age, overall health of the patient, the type offormulation, the mode or manner or administration, or the presence orabsence of risk factors that significantly increase the likelihood thatthe breast cancer will recur in the patient. Such risk factors include,but are not limited to the type of surgery, status of lymph nodes andthe number positive, the size of the tumor, the histologic grade of thetumor, the presence/absence of hormone receptors (estrogen andprogesterone receptors), HER2/neu expression, lymphovascular invasion,and genetic predisposition (BRCA 1 and 2). In some preferred aspects,the effective amount is dependent on whether the patient is lymph nodepositive of lymph node negative, and if the patient is lymph nodepositive, the number and extent of the positive nodes. In all cases, theappropriate effective amount can be routinely determined by those ofskill in the art using routine optimization techniques and the skilledand informed judgment of the practitioner and other factors evident tothose skilled in the art. Preferably, a therapeutically effective doseof the vaccine compositions described herein will provide thetherapeutic preventive benefit without causing substantial toxicity tothe subject.

Toxicity and therapeutic efficacy of the vaccine compositions can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Vaccine compositions that exhibit large therapeutic indicesare preferred. Data obtained from cell culture assays and animal studiescan be used in formulating a range of dosage for use in patients. Thedosage of such vaccine compositions lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized.

Toxicity information can be used to more accurately determine usefuldoses in a specified subject such as a human. The treating physician canterminate, interrupt, or adjust administration due to toxicity, or toorgan dysfunctions, and can adjust treatment as necessary if theclinical response is not adequate, to improve the response. Themagnitude of an administrated dose in the prevention of recurrent breastcancer will vary with the severity of the patient's condition, relativerisk for recurrence, or the route of administration, among otherfactors. The severity of the patient's condition can, for example, beevaluated, in part, by standard prognostic evaluation methods.

The vaccine compositions can be administered to a patient on anyschedule appropriate to induce and/or sustain protective immunityagainst breast cancer relapse, and more specifically to induce and/orsustain a cytotoxic T lymphocyte response to E75 (SEQ ID NO:2). Forexample, patients can be administered a vaccine composition as a primaryimmunization as described and exemplified herein, followed byadministration of a booster to bolster and/or maintain the protectiveimmunity.

In some aspects, patients can be administered the vaccine compositions1, 2 or more times per month. Once per month for six consecutive monthsis preferred to establish the protective immune response, particularlywith respect to the primary immunization schedule. In some aspects,boosters can be administered at regular intervals such as every 6 ormore months after completion of the primary immunization schedule.Administration of the booster is preferably every 6 months. Boosters canalso be administered on an as-needed basis.

The vaccine administration schedule, including primary immunization andbooster administration, can continue as long as needed for the patient,for example, over the course of several years, to over the lifetime ofthe patient. In some aspects, the vaccine schedule includes morefrequent administration at the beginning of the vaccine regimen, andincludes less frequent administration (e.g., boosters) over time tomaintain the protective immunity.

The vaccine can be administered at lower doses at the beginning of thevaccine regimen, with higher doses administered over time. The vaccinescan also be administered at higher doses at the beginning of the vaccineregimen, with lower doses administered over time. The frequency ofprimary vaccine and booster administration and dose of E75 administeredcan be tailored and/or adjusted to meet the particular needs ofindividual patients, as determined by the administering physicianaccording to any means suitable in the art.

In some aspects, the vaccine compositions, including compositions foradministration as a booster, comprise from about 0.1 mg to about 10 mgof E75 peptide. In some preferred aspects, the compositions compriseabout 0.5 mg of E75. In some preferred aspects, the compositionscomprise about 2 mg of E75. In some preferred aspects, the compositionscomprise about 1 mg of E75.

In some preferred aspects, the vaccine compositions comprising E75,including compositions for administration as a booster, further compriseGM-CSF. Such compositions preferably comprise from about 0.01 mg toabout 0.5 mg of GM-CSF. In some preferred aspects, the compositionscomprise about 0.125 mg of GM-CSF. In some preferred aspects, thecompositions comprise about 0.25 mg of GM-CSF.

In some particularly preferred aspects, the vaccine compositionscomprise 1 mg of E75 peptide and from 0.125 to 0.250 mg of GM-CSF in atotal volume of 1 ml, and are administered monthly as a splitinoculation of 0.5 ml each, administered by injections about 5 cm aparton the patient's body, and administered concurrently or admixed. Theadministration schedule is preferably monthly for six months. After aperiod of about 48 hours, the injection site can be assessed for localreaction of erythema and induration. If the reactions at both sites areconfluent and the area of total induration measures >100 mm (or thepatient experiences any >grade 2 systemic toxicity), then the dose ofGM-CSF can be reduced, for example, by half, though it is intended thatthe peptide dose remain the same. If the patient presents a robustreaction on subsequent doses, then further reduction of GM-CSF canoccur, for example, reducing by half. If the patient does not presentwith a robust reaction, then the patient can continue with the higherGM-CSF dose. In some aspects, the administration schedule and dosing ofthe booster is similarly determined, with boosters beginning withadministration of vaccine compositions comprising 1 mg of E75 and 0.25mg GM-CSF, administered about every six months following the conclusionof the primary immunization vaccine schedule.

The following Exemplary Aspects of specific examples for carrying outthe present invention are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Example 1 Patient Selection

The node-positive (NP) and node-negative (NN) trials were approved bythe local Institutional Review Boards and conducted at Walter Reed ArmyMedical Center, Washington, D.C. and the Joyce Murtha Breast CareCenter, Windber, Pa. under an investigational new drug application(BB-IND#9187). All patients had histologically confirmed breast cancer(BCa), and completed a standard course of surgery, chemotherapy, andradiation therapy (as required) before enrollment. Patients on hormonaltherapy were continued on their specific regimen. After propercounseling and consenting, BCa patients were enrolled to the appropriatetrial (NP or NN) and then HLA typed, since E75 binds primarily HLA-A2found in approximately 40-50% of the general population. HLA-A2⁺patients were vaccinated, and HLA-A2⁻ patients were observedprospectively for clinical recurrence. HLA-A3⁺ patients were enrolledinto a parallel trial with the A2 patients, and treated on the activedose schedule at the time of enrollment. Before vaccination, patientswere skin tested with a panel of recall antigens (mumps, tetanus, andCandida). Patients were considered immunocompetent if they reacted (>5mm) to >2 antigens.

A total of 186 patients were enrolled in both E75 vaccine trials (NP=95,NN=91), who were disease-free after standard therapy, but at high riskfor recurrence. After HLA-A2⁺, and later HLA-A3⁺, patients (n=101) werevaccinated (49 NP and 52 NN; 90 HLA-A2⁺ and 11 HLA-A3⁺). All otherpatients (n=85) were assigned to observation. Five vaccine and fourobservation patients withdrew from the study, though none due totoxicity. Therefore, 96 vaccinated patients and 81 observation patientswere available for analysis. Demographics and prognostic factors forboth groups are presented in Table 1.

TABLE 1 Demographic and prognostic factors for vaccinated andobservation patients. Vaccinated, Observed, HLA-A2⁺, A3⁺ HLA-A2⁻, A3⁻ (n= 96)^(†) (n = 81)^(‡) P Median age, years 58.9 55.1 Range, years 32-8034-87 0.33 Race White, % 89.6 81.5 Other, % 10.4 18.5 0.12 Tumor sizeT1, % 69.8 60.5 0.20 T2-T4, % 30.2 39.5 0.20 Histological grade I-II, %64.5 59.5 0.50 III, % 35.5 40.5 0.50 Node-positive, % 46.9 56.8 0.19Median + nodes  2.0  2.5 (NP only) Range  1-25  1-15 0.17 HER2/neu IHC3+ or 25.8 28.4 0.32 FISH+, % Hormone receptor 31.6 17.3 0.03 negative,% XRT, % 71.9 80.2 0.20 Chemoprevention, % 65.6 78.8 0.05 AdjuvantHerceptin, %  5.2  3.7 0.60 ^(†)101 patients enrolled to vaccine arm, 2switched to observation, 1 withdrew for adjuvant trastuzumab, 1 due toan extended unrelated illness, and 1 patient for personal reasons.^(‡)85 patients enrolled to observation arm, 2 lost to follow-up and 4withdrew for our MHC II peptide vaccine trial. Two patients were gainedfrom the vaccine arm.

The two groups were equivalent in most standard prognostic categories.However, more vaccinated patients were hormone-receptor negative, and,therefore, fewer patients in the vaccine group were on adjuvant hormonaltherapy. In looking at the individual trials, more vaccinated patientsin the NN trial compared to controls had HER2/neu over-expressing tumors(25.0% vs. 7.1%, P<0.05), and fewer received adjuvant radiotherapy(64.7% vs. 85.7%, P<0.05).

During the trials, it was determined that E75 could be used in HLA-A3⁺patients based on binding affinity data obtained from two commonly usedHLA-peptide binding algorithms: BIMAS (SEQ ID NO:3) and SYFPEITHI (SEQID NO:4). Additionally, pre-clinical evaluation demonstrated thatE75-stimulated HLA-A3⁺ CTL could lyse HLA-A3⁺ HER2/neu-expressing cancercells (not shown).

Although there was no difference in the nodal status of the HLA-A3subset compared to the HLA-A2 subset (54.5% vs. 45.9%, P=0.59), theytended to have smaller tumors (90.9% T1 vs. 65.7%, P=0.08), were lesslikely to have hormonally insensitive tumors (18.2% vs. 29.6%, P=0.4),and had less HER2/neu overexpressing tumors (0% vs. 31.5%, P=0.028).

Example 2 Vaccination and Clinical Protocol

The E75 peptide was commercially produced in good manufacturingpractices grade by NeoMPS, Inc. (San Diego, Calif.). Peptide purity(>95%) was verified by high-performance liquid chromatography and massspectrometry, and the amino acid content was determined by amino acidanalysis. Sterility and general safety testing was carried out by themanufacturer. Lyophilized peptide was reconstituted in sterile saline at100 μg, 500 μg, or 1000 μg in 0.5 ml. At the time of administration, thepeptide was thawed and mixed with GM-CSF (Berlex, Seattle, Wash.) in 0.5ml, and the 1.0 ml inoculation was split and given intradermally at twosites 5 cm apart. All inoculations were given in the same extremity.

Vaccination Series. The NP trial was designed as a two stage safetytrial with escalating doses of peptide in the initial stage andalterations of schedule in the latter stage. Details of the vaccineseries have been previously published (Peoples G E et al. (2005) J.Clin. Oncol. 23:7536-45). Briefly, 3-6 patients (HLA-A2⁺ or HLA-A3⁺)were each assigned to receive four or six monthly injections of 100 μg,500 μg, or 1000 μg of E75 (100.6, 500.4, 500.6, 1000.4 and 1000.6,respectively) (Table 2). Groups were ultimately expanded in order todetermine and confirm optimal dosing in NP patients, accounting for thelarger number of patients in the latter dose groups.

TABLE 2 NP and NN trial designs. No. of patients Peptide GM-CSF PatientHLA-A2⁺ dose^(†) dose^(†) Months Group (A3⁺) (μg) (μg) vaccinated^(‡)Node-positive 100.6  2* 100 250 0, 1, 2, 3, 4, 5 500.4 6 500 250 0, 1,2, 5 500.6 6 500 250 0, 1, 2, 3, 4, 5 1000.4  9 (2) 1000 250 0, 1, 2, 51000.6 16 (4) 1000 250 0, 1, 2, 3, 4, 5 Node-negative 500.125.3 10  500125 0, 1, 5 500.125.4 10  500 125 0, 1, 2, 5 500.250.4 10 (3) 500 250 0,1, 2, 5 500.250.6 10 (2) 1000 250 0, 1, 2, 3, 4, 5 1000.250.6  6 1000250 0, 1, 2, 3, 4, 5 Total 85 (11) ^(†)Peptide was suspended in 0.5 mlsterile saline and combined with GM-CSF and sterile saline to finalvolume of 1.0 ml per inoculation. ^(‡)Vaccines were administered every3-4 weeks. *One patient assigned to 100.6 group withdrew and noreplacement at that dose group was designated.

The NN trial was designed to further delineate optimal biologic dosingby varying the dose of GM-CSF and altering the inoculation schedule.Patients with non-HER2/neu-expres sing tumors were allowed in this trialto determine the feasibility of vaccinating a presumably antigen-naïvehost. Ten patients were assigned to each dose group to receive three,four, or six monthly injections over five months (Table 2).

Peripheral Blood Mononuclear Cell (PBMC) Isolation and Cultures.

Blood was drawn before each vaccination and at one (post-vaccine) andsix months (long-term) after vaccine series completion. 50 ml of bloodwas drawn and PBMCs were isolated. PBMCs were washed and re-suspended inculture medium and used as a source of lymphocytes.

Toxicity.

Patients were observed one hour post-vaccination for immediatehypersensitivity and returned 48-72 hours later to have their injectionsites measured and questioned about toxicities. Toxicities were gradedby the NCI Common Terminology Criteria for Adverse Events, v3.0 andreported on a scale from 0-5. Progression from one dose group to thenext occurred only if no significant toxicity occurred in the lower dosegroup. Patient-specific results were reported based on maximal local andsystemic toxicity occurring during the series.

Local and systemic toxicities were mild, and all patients completed thevaccine series. Local toxicities were grade 1 (81%) and grade 2 (19%).Systemic toxicity was minimal: grade 0 (12%), grade 1 (71%), grade 2(14%) and grade 3 (2%) (FIG. 1) with no grade 4 or 5 systemic toxicitiesobserved. Since toxicities observed are consistent with GM-CSF, a 50%dose reduction in GM-CSF was instituted in the event of significantlocal or systemic reactions (18.7% of patients).

Toxicity profiles were the same in A3 patients as their A2 counterparts:maximum local toxicity grade 1 (82%) and grade 2 (18%) for both groups.Maximum systemic toxicity (A3 vs. A2): grade 0 (0% vs. 15%), grade 1(92% vs. 68%), grade 2 (8% vs. 14%) and grade 3 (0% vs. 2%; p=0.4).Local responses of A3 patients were identical to the A2 patients withinthe respective dose groups. Thus, there was no difference in thetoxicity profile among the HLA-A3⁺ patients compared to the HLA-A2⁺patients, and the local reactions were just as robust. Grade 2 localtoxicity was 20% compared to 18%, respectively, suggesting similar invivo immunogenicity.

Clinical Recurrences.

All patients were observed for clinical recurrence per standard of carecancer screening as dictated by the patient's primary oncologist. Apatient was considered recurrent if biopsy proven or if treated forrecurrence by the primary oncology team.

Per protocol design, primary analysis was initiated at 18 months medianfollow-up. At completion of this analysis, 171 patients had beenenrolled, and the recurrence rate in the vaccinated group was 5.6%compared to 14.2% in the observation group (P=0.04) at a medianfollow-up of 20 months. The disease-free survival rates in thevaccinated and control groups were 92.5% and 77%, respectively (FIG. 2).There were four deaths in the observation group (overall survival [OS]95.1%) compared to only one death in the vaccinated group (OS 99%,P=0.1).

The follow-up of both trials was extended to five years despite waningimmunity and lack of a booster inoculation in the protocol design. Anupdated analysis documented additional recurrences in both groupsincluding a late recurrence in the vaccine group at 58 months. At amedian follow-up of 26 months, there were 186 patients enrolled, and therecurrence rate was 8.3% in the vaccine group compared to 14.8% in theobservation group (P=0.15). There was a different distribution ofrecurrences among these patients. Bone only recurrence accounted for 50%of the recurrences in the control patients (6/12) and 0% of thevaccinated recurrent patients (P=0.04).

Among the HLA A3⁺ patients, the recurrence rate was similar to theHLA-A2⁺ patients (9.1% vs. 8.2%).

Statistical Analysis.

Recurrence rates were compared between groups using survival analysis bythe Kaplan-Meier method, and the proportion of subjects who hadrecurrences compared using log-ranked analysis. P values forclinico-pathologic factors were calculated using Wilcoxon, Fisher'sexact test or χ² as appropriate. P values for comparing pre-vaccinationand post-vaccination dimer levels were calculated using Wilcoxon and forDTH using Student's t-test.

Example 3 HLA-A2:Immunoglobulin Dimer Assay

The presence of CD8⁺E75-specific cells in freshly isolated PBMC frompatients was directly assessed by using a dimer assay. In brief, theHLA-A2:immunoglobulin (Ig) dimer (PharMingen, San Diego, Calif.) wasloaded with the E75 or control peptide (E37, folate binding protein(25-33) RIAWARTEL (SEQ ID NO:5)) by incubating 1 μg of dimer with anexcess (5 μg) of peptide and 0.5 μg of β2-microglobulin (Sigma™, St.Louis, Mo.) at 37° C. overnight then stored at 4° C. until used. PBMCwere washed and re-suspended in PharMingen Stain Buffer (PharMingen) andadded at 5×10⁵ cells/100 μl/tube in 5 ml round-bottom polystyrene tubes(Becton Dickinson, Mountain View, Calif.) and stained with the loadeddimers and antibodies. In each patient the level of CD8⁺E75-specificcells was determined in response to each successive vaccination and allpost-inoculation measurements were averaged for each patient andcompared with their pre-inoculation levels.

E75-specific CTL were assessed in fresh ex vivo PMBCs by the dimer assaybefore each vaccination and at one (post-vaccination) and six months(long-term). The dimer assay has been previously shown to correlate withfunctional immune assays (cytotoxicity and cytokine secretion) (PeoplesG E et al. (2005) J. Clin. Oncol. 23:7536-45). A pattern of increasingCD8⁺E75-specific CTL was observed during the vaccine series, peaking andthen receding to a plateau by completion.

The cumulative dimer responses for all patients are shown in FIG. 3A.There was a statistically significant increase in the medianCD8⁺E75-specific cells from pre-vaccine to post-vaccination and to peaklevels. Long-term levels were not different from the pre-vaccinationlevels. Only 48.3% of patients maintained significant residual immunity(defined as dimer >0.5) six months post-vaccination.

Pre-existing immunity to E75 (defined as dimer >0.3) was found in 42.7%of patients (FIG. 3B). The same pattern of dimer response was seenregardless of initial dimer levels. However, patients who lackedpre-existing immunity had a significant increase in their long-termdimer levels.

Example 4 Delayed Type Hypersensitivity

In both trials, a DTH reaction was assessed with 100 μg of E75 in 0.5 mlof normal saline (without GM-CSF) and 0.5 ml normal saline as a volumecontrol one month after completion of the vaccine series as describedpreviously (Peoples G E et al. (2005) J. Clin. Oncol. 23:7536-45). TheDTH reaction was measured in two dimensions at 48-72 hours by using thesensitive ballpoint-pen method and reported as the orthogonal mean andcompared to control. In the NN trial, a DTH was also performedpre-vaccination.

In Vivo Immune Response.

To measure the vaccine's in vivo effectiveness, a post-vaccine DTH wasmeasured one month after vaccine series completion with 100 μg of E75injected intradermally with a saline volume control. Among allvaccinated patients, 74% had a positive post-vaccine DTH with an averageinduration to E75 of 14.0±1.4 mm compared to control 2.1±0.5 mm(P<0.0001) (FIG. 4A).

NN patients had a pre-vaccine DTH as well as post-vaccine DTH (FIG. 4B).Pre-vaccination, there was no difference in DTH between E75 and control.Post-vaccination, the DTH response to E75 was statistically larger thancontrol, and the E75 DTH was significantly different post-vaccinecompared to pre-vaccine (10.9±1.5 mm vs. 2.8±0.8 mm, P<0.0001).

NP patients had a larger post-vaccination E75 DTH response than NNpatients (FIG. 4C); a difference likely due to the NN patients receivingmuch lower amounts of E75 overall. Assessing DTH responses as a functionof dose, those patients receiving 6000 μg of E75 had a significantlylarger DTH reaction compared with those patients receiving <6000 μg ofpeptide (25.1±4.0 mm vs. 13.3±1.9 mm, P=0.008) (FIG. 4D).

The HLA-A3⁺ patients had comparable post-vaccination DTH to the HLA-A2+(10.5±2.7 mm vs. 15.1±1.9 mm, P=0.38).

Example 5 HLA-A3⁺ ELISPOT Assay

Vaccine response for HLA-A3⁺ patients was also assessed by E75-specificinterferon-γ ELISPOT. By ELISPOT, the A3 patients demonstrated a rangeof 0-30 spots/10⁶ cells at baseline that increased to a range of 3-448spots/10⁶ cells post-vaccination, p=0.04. Most importantly, clinicalrecurrences were the same in both groups (A3, 9.1% vs A2, 8.2%) andcompared to 14.8% in the control group.

Example 6 Breast Cancer Vaccine Booster

Patients.

The NP and NN trials were approved at the local Institutional ReviewBoards and conducted at Walter Reed Army Medical Center (WRAMC),Washington D.C. and the Joyce Murtha Breast Care Center, Windber, Pa.These clinical trials are being conducted under an investigational newdrug application (BB-TND #9187) approved by the Food and DrugAdministration. All patients had histologically confirmed breast cancer,had completed standard therapy, were disease-free and immunocompetent attime of initial enrollment. HLA-A2⁺ and HLA-A3⁺ patients were vaccinatedwith varying doses of E75 and GM-CSF and on varying schedules over a sixmonth period, as set forth in the Examples above. Patients were offeredan optional booster dose of E75 (1 mg)+GM-CSF (0.250 mg) if they were atleast six months from completion of their primary vaccination series.

25 patients received a booster vaccination (Table 3). Just over half(56%) had NP breast cancer. The median time from prior vaccination was12 months (range 6-24 months). Patients were evaluated as either earlybooster (EB) patients if they received the booster 6 months afterprimary series or late booster patients (LB) if they were >6 months fromprimary series.

TABLE 3 Patient Demographics. Patients (n = 25) Age, median (yrs) 56(range 31-76) ≧T2 28% Node positive 56% Grade 3 32% ER-PR- 28% HER2/neuoverexpression 20% Time from primary standard 33 (9-200) therapy (mos)Time from primary vaccine 12 (6-24) series (mos)

Residual E75-specific immunity declined over time as measured byHLA-A2:IgG dimer. The median level of CD8+ T-cells in EB group (n=6) was1.4% (0.61-3.43% range) compared to the LB group (n=13) (0.44%, 0-2.67%,p=0.02). For the LB patients, their median dimer level 6 months afterthe initial series was 0.70% (0.19%-1.55%). This was not statisticallydifferent from EB patients' 6 month dimer levels (FIG. 5).

Booster Vaccine.

The E75 peptide was commercially produced in good manufacturingpractices grade by NeoMPS (San Diego, Calif.). Peptide purity wasverified by high-performance liquid chromatography and massspectrometry, and the amino acid content was determined by amino acidanalysis. The peptide was purified to more than 95%. Sterility andgeneral safety testing was carried out by the manufacturer. Lyophilizedpeptide was reconstituted in sterile saline at a concentration of 1000μg in 0.5 mL. The peptide was mixed with GM-CSF (Berlex, Seattle, Wash.)in 0.5 mL, and the 1.0 mL inoculation was split and given intradermallyat two sites 5 cm apart. Booster vaccination was given in the sameextremity as the primary series.

Toxicity.

Patients were observed 1 hour post vaccination for immediatehypersensitivity reactions. Toxicities were graded by the NCI CommonToxicity Criteria for Adverse Events, v3.0 and reported on a scale from0 to 5. Patients who had previously had significant (grade 2 or 3) localor systemic toxicity received a reduced dose of GM-CSF at 0.125 mg.

The booster dose was very well tolerated (FIG. 6) with primarily grade 1local toxicity (a desired effect). Over half of the patients had nosystemic complaints. There were no grade 3 or 4 toxicities. Only 1patient (4%) had a higher grade toxicity during the booster than duringthe primary series (grade 2 local inflammation).

Peripheral Blood Mononuclear Cell Isolation and Cultures.

Blood was drawn before booster vaccination, and 3 to 4 weeks followingbooster administration to isolate peripheral blood mononuclear cells inVacutainer CPT tubes, and used as a source of lymphocytes.

HLA-A2:Immunoglobulin Dimer Assay.

The presence of CD8⁺E75-specific cells in freshly isolated PBMCs frompatients was directly assessed by using the dimer assay described inExample 3 above. Briefly, the HLA-A2:immunoglobulin (Ig) dimer(Pharmingen, San Diego, Calif.) was loaded with the E75 or controlpeptide (folate binding protein peptide-E37 (25-33) RIAWARTEL (SEQ IDNO:5)) by incubating 1 μg of dimer with an excess (5 μg) of peptide and0.5 μg of β2-microglobulin (Sigma Chemical Co, St Louis, Mo.) at 37° C.overnight then stored at 4° C. until used. PBMC were washed andre-suspended in PharMingen Stain Buffer (Pharmingen, San Diego, Calif.)and were added at 5×10⁵ cells/100 μl/tube in 5 ml round-bottompolystyrene tubes (Becton Dickinson, Mountain View, Calif.) and stainedwith the loaded dimers and antibodies. In each HLA-A2⁺ patient, thelevel of CD8⁺E75-specific cells was determined before and after thevaccine booster.

Antigen specific CD8⁺ T-cells were quantified before and 3-4 weeks afterbooster vaccination. Significant residual immunity (SRI, defined asantigen specific CD8⁺ T-cells ≧0.5%) was significantly different in thetwo groups at 100% (6/6) in the EB patients compared to 30.8% (4/13) ofLB patients (p=0.01). Among those patients lacking SRI (n=8) there was atrend towards increased E75-specific CD8+ T-cells (FIG. 7) from 0.43%(0-0.49%) to 0.87% (0-2.3%; p=0.08).

Enzyme-Linked Immunospot Assay.

IFN-γ producing cells were detected using the BD ELISPOT kit eitherimmediately (ex vivo) or after 7-day incubation with peptides. FreshPBMC were plated into the ELISPOT plate at a concentration of 5×10⁵cells (ex vivo) or 1×10⁵ (7-day) per well in medium containing IL-7 (exvivo) or in medium with and without IL-7 (7-day). Cells were stimulatedfor 16 hours (ex vivo) or 7 days in the presence or absence of peptides(E37, FluM, E75, GP2, HER2/neu 1 μg or 5 μg). Additional incubations inthe 7-day wells included combination of E75+HER2/neu 1 μg or 5 μg. Atotal of 16 assays were performed on each blood sample, provided enoughcells were available. At the end of incubation, the plates weredeveloped as per manufacturer's instructions. A biotinylated detectionantibody was added, and the plates incubated overnight at 4° C.Following incubation, the plates were washed, Avidin-HRP solution wasadded for 1 hour, and spots developed using AEC substrate solution.Spots were counted using the Immunospot Series 2 analyzer and ImmunoSpotsoftware.

All patients had IFN-γ producing cells quantified before and afterbooster vaccination in up to 16 different assays, depending onavailability of PBMC from a single blood draw. Twenty two patients hadat least one paired pre- and post-booster ELISPOT assay (median 10assays per patient, range 1-14). Among these patients, there were 255total assays pre-booster, of which 54.5% showed detectable IFN-γproducing. Among 194 paired assays (pre- and post-booster samples fromsame patient run with same peptide concentration), 78 (40.2%) showedincreased IFN-γ producing cells with booster. In all, 20/22 (91%) ofpatients showed increased IFN-γ producing cells in at least one assayand 11 (50%) showed increased IFN-γ producing cells in at least 50% ofthe assays. Results are shown in FIG. 8.

Local Reactions.

Local reactions (LR) were measured as an in vivo functional assessmentof response. LR were measured 48-72 hours post-vaccination and measuredin two directions and reported as an orthogonal mean±SE using thesensitive ball point method. LR were compared to the patient's ownprevious LR to assess response to booster.

Patients who received the booster ≦9 months (n=12) from their primaryseries had significantly larger LR (103±7 mm) than patients >9 months(n=13) from primary series (79±4 mm, p=0.01). There was no difference inthe two groups when comparing them at the end of the primary series (≦9months 81±5 mm; >9 months 85±8 mm, p=0.73). Results are shown in FIGS. 9and 10.

Statistical Analysis.

HLA:IgG dimer values are reported as medians and P values werecalculated using the Wilcoxon test. For proportional comparisons,Fisher's exact test was used. Comparison of local reactions was madewith paired or unpaired Student's t-test, as appropriate.

Example 7 HER2/neu (E75) Peptide Vaccine Response per HER2/neuExpression Level

Clinical trials were conducted with the HER2/neu E75-peptide vaccine innode-positive and node-negative BCa patients. These patients consist ofall levels of HER2/neu expression. Determining HER2/neu status isperformed predominately via two tests, immunohistochemistry (IHC) andfluorescence in situ hybridization (FISH). IHC detects over-expressionof HER2/neu protein and is reported on a semi-quantitative scale of 0 to3+ (0=negative, 1+=low expression, 2+=intermediate, and3+=over-expression). FISH on the other hand detects amplification(excess copies) of the HER2/neu gene and is expressed as a ratio ofHER2/neu to chromosome 17 and interpreted as over-expression if FISHis >2.0 copies. Concurrence rate of IHC and FISH is approximately 90%.

Materials and Methods:

A subset analysis was performed of 163 BCa patients enrolled in ourphase II E75 vaccine trials based on level of HER2/neu expression.Patients were assessed low-expressors (LE=IHC 1+-2+ and FISH >0 but<2.0) vs. over-expressors (OE=IHC 3+ and/or FISH >2.0), and by IHCstatus (0, 1+, 2+, 3+). Analysis was performed of standardclinocopathlogic factors, immunologic response to the vaccine (in vivoDTH reactions and in vitro HLA-A2:IgG dimer assay), and clinicalresponses (absolute recurrence and mortality rates).

Patient Characteristics and Clinical Protocols:

The E75 NP and NN trials were approved by the Institutional ReviewBoards and conducted at Walter Reed Army Medical Center, Washington,D.C. and the Joyce Murtha Breast Care Center, Windber, Pa. underinvestigational new drug application (BB-IND#9187). All patients hadhistologically confirmed BCa, and had completed a standard course ofsurgery, chemotherapy, and radiation (as required) before enrollment.Patients on hormonal therapy were continued on their regimen. Afterproper counseling and consenting, BCa patients were enrolled to theappropriate trial (NP or NN) and HLA typed since E75 binds primarilyHLA-A2 found in approximately 40-50% of the general population. HLA-A2+patients were vaccinated, and HLA-A2− patients were observedprospectively for clinical recurrence. Subsequently HLA-A3+ patientswere vaccinated. Before vaccination, patients were skin tested with apanel of recall antigens (Mantoux test). Patients were consideredimmunocompetent if they reacted (>5 mm) to >2 antigens.

Vaccine:

The E75 peptide was commercially produced in good manufacturingpractices grade by NeoMPS, Inc. (San Diego, Calif.). Peptide purity(>95%) was verified by high-performance liquid chromatography and massspectrometry. Sterility and general safety testing was carried out bythe manufacturer. Lyophilized peptide was reconstituted in 0.5 mlsterile saline at 100 mcg, 500 mcg, or 1000 mcg. The peptide was mixedwith GM-CSF (Berlex, Seattle, Wash.) in 0.5 ml. The 1.0 ml inoculationwas split and given intradermally at two sites 5 cm apart in the sameextremity.

Vaccination Series:

The NP trial was designed as a two stage safety trial with escalatingdoses of peptide in the initial stage and alterations of schedule in thelatter stage. Details of the vaccine series have been previouslypublished. Briefly, 3-6 patients were each assigned to receive four orsix monthly injections of 100 mcg, 500 mcg, or 1000 mcg of E75 peptide(100:6, 500:4, 500:6, 1000:4 and 1000:6, respectively). Groups wereultimately expanded in order to determine and confirm optimal dosing inNP patients, accounting for the larger number of patients in the latterdose groups.

The NN trial was designed to further delineate optimal biologic dose byvarying the dose of GM-CSF and altering the inoculation schedule. Twelvepatients with HER2/neu IHC 0 tumors were allowed in this trial todetermine the feasibility of vaccinating a presumably antigen-naïvehost. Ten patients were assigned to each dose group with constant E75peptide of 500 mcg to receive three, four, or six monthly injectionswith varying GM-CSF doses (125 mcg or 250 mcg).

Toxicity:

Patients were observed one hour post-vaccination for immediatehypersensitivity and returned 48-72 hours later to have their injectionsites measured and questioned about toxicities. Toxicities were gradedby the NCI Common Terminology Criteria for Adverse Events v3.0 (reportedon 0-5 scale). Progression from one dose group to the next occurred onlyif no significant toxicity occurred in the lower dose group.Patient-specific results are reported based on maximal local andsystemic toxicity occurring during the series.

Peripheral Blood Mononuclear Cell (PBMC) Isolation and Cultures:

Blood was drawn before each vaccination and at one (post-vaccine) andsix months (long-term) after vaccine series completion. 50 ml of bloodwas drawn and PBMCs isolated. PBMCs were washed and re-suspended inculture medium and used as a source of lymphocytes as previouslydescribed.

HLA-A2:Immunoglobulin Dimer Assay:

The presence of CD8+E75-specific cells in freshly isolated PBMCs frompatients was directly assessed by using the dimer assay as previouslydescribed. Briefly, the HLA-A2:immunoglobulin (Ig) dimer (PharMingen,San Diego, Calif.) was loaded with the E75 or control peptide (E37,folate binding protein (25-33) RIAWARTEL (SEQ ID NO:5)) by incubating 1mcg of dimer with an excess (5 mcg) of peptide and 0.5 mcg ofβ2-microglobulin (Sigma, St. Louis, Mo.) at 37° C. overnight then storedat 4° C. until used. PBMCs were washed and re-suspended in PharMingenStain Buffer (PharMingen) and added at 5×10⁵ cells/100 μl/tube in 5 mlround-bottom polystyrene tubes (Becton Dickinson, Mountain View, Calif.)and stained with the loaded dimers and antibodies. In each patient thelevel of CD8+E75-specific cells was determined in response to eachsuccessive vaccination, and average post-inoculation levels werecompared to their pre-inoculation levels.

Delayed Type Hypersensitivity (DTH):

In both trials, a DTH reaction was assessed with 100 mcg of E75 peptidein 0.5 ml of normal saline (without GM-CSF) and 0.5 ml normal saline asa volume control one month after completion of the vaccine series asdescribed previously. The DTH reaction was measured in two dimensions at48-72 hours by using the sensitive ballpoint-pen method and reported asthe orthogonal mean and compared to control. In the NN trial, a DTH wasalso performed pre-vaccination as well.

Clinical Recurrences:

All patients were observed for clinical recurrence per standard of carecancer screening as dictated by the patient's primary oncologist. Apatient was considered recurrent if biopsy proven or if treated forrecurrence by the primary oncology team.

Statistical Analysis:

Recurrence rates were compared between groups using survival analysis bythe Kaplan-Meier method, and the proportion of subjects who hadrecurrences compared using log-ranked analysis. P values forclinico-pathologic factors were calculated using Wilcoxon, Fisher'sexact test or χ2 as appropriate. P values for comparing pre- andpost-vaccine dimer levels and DTH were calculated using paired orunpaired two-tailed Student t-test.

Results:

LE (control=44, vaccine=56) vs. OE patients (control=22, vaccine=29) andIHC status control and vaccine groups (0=5 vs. 7, 1+15 vs. 25, 2+24 vs.26, 3+13 vs. 19 respectively) were assessed. Both LE vs. OE and all IHCstatus vaccinated groups responded immunologically; however LE patients,and more specifically IHC 1+ patients, had increased long-term in vitroimmune response (p=0.04 and p=0.08 respectively). In addition, LEpatients trended towards, and IHC 1+ patients had, decreased mortalitycompared to their control groups (p=0.08 and p=0.04 respectively).

Patients:

186 patients were enrolled in the E75 vaccine studies; 9 withdrew (4control patients and 5 vaccinated patients—none withdrew due totoxicity) resulting in 177 completing the trials. All control (C) andvaccinated (V) patients in the NP trial (C=46, V=45, total=91 patients)had IHC, FISH, or both tests performed. In the NN trial (C=35, V=51,total=86 patients) 12 patients had HER2/neu IHC 0 tumors (C=5, V=7).Also in the NN trial, 14 patient's (C=7, V=7) tumors did not undergo IHCor FISH—these 14 patients have been excluded from subset analysis;therefore 163 patients were available for analysis.

LE vs. OE Subset Analysis:

Patients per Expression:

Subset analysis was performed comparing LE (IHC 1+-2+ or FISH >0 and<2.0) vs. OE (IHC 3+ or FISH >2.0). Sixty-six patients in the controlgroup had IHC or FISH performed (LE=44, OE=22). A total of 85 patientsin the E75 vaccine group had IHC or FISH performed (LE=56, OE=29). Acomparable number of C and V patients were in the LE (67% vs. 66%respectively) and OE groups (33% vs. 34% respectively).

Demographics, prognostic factors, and treatment profiles of LE vs. OEpatients are presented in Table 4. In regards to LE patients nostatistical differences were noted between C and V patients. With OEpatients, a statistically larger number of V patients were hormonereceptor negative than in the C group (p=0.02) (Table 4).

TABLE 4 Demographics, prognostic factors, and treatment profiles ofpatients enrolled in E75 Phase II trial by LE vs. OE. LE LE OE OEControl Vaccine Control Vaccine (n = 44) (n = 56) P (n = 22) (n = 29) PMedian age, 55 56 50 52 years Range years 31-82 27-77 0.7 32-75 37-680.1 Race White, % 86.4% 89.3% 0.8 72.7% 86.2% 0.3 Other, % 13.6% 10.7%0.8 27.3% 13.8% 0.3 Tumor size T2-T4, % 38.6% 33.9% 0.7 31.8% 34.5% 0.9Histological grade Grade III, % 27.2% 30.4% 0.8 63.6% 62.1% 0.9 NodePositive 54.5% 58.9% 0.7 90.1% 55.2%  0.06 (NP), % Hormone 15.9% 19.6%0.8 27.3% 62.1%  0.02* receptor-, % Chemotherapy, 72.7% 75.0% 0.8 86.4%96.6% 0.3 % XRT, % 84.1% 75.0% 0.3 72.7% 75.9% NS Hormonal 81.8% 76.8%0.6 63.6% 41.4% 0.2 therapy, % Herceptin, %  0.2%  0.2% NS  9.1% 24.1%0.3 *Statistically significant difference.

Immunologic Response per Expression:

The E75 vaccine was capable of eliciting an in vitro immune response inboth the LE and OE patients. Significant increases from pre-vaccine tomax E75-specific CD8+ T cells was noted in both groups (LE p<0.001, OEp<0.001). LE patients had statistically higher maximum immune responsecompared to OE patients (p=0.04) (FIG. 11A).

Both LE and OE patients were able to elicit an in vivo immune responseas measured via DTH pre and post-vaccine. Significant pre-post DTHincreases were noted in both categories (LE p<0.001, OE p=0.02) (FIG.11B). Although the LE post-DTH is larger than OE post-DTH (15.9+1.9 mmvs. 12.8+2.0 mm, respectively), there is no statistical significance(p=0.5). Overall, the E75 vaccine appears more immunologically active inLE patients.

Clinical Response per Expression:

Clinical response, evaluated by recurrence and mortality, is noted inFIGS. 11C and 11D. All V patients (LE=10.7% vs. OE=13.8%) appeared tohave decreased recurrence rates when compared to the C patients (LE &OE=18.2%), but these numbers were not statistically significant. Moreimportantly, there was a trend towards decreased mortality in the Vpatients, most impressively seen in the LE patients (C=6.8% vs. V=0.0%;p=0.08).

IHC Status Subset Analysis:

Patients per IHC Status:

The C group had 57 patients' pathology specimens that underwent IHC(0=5, 1+=15, 2+=24, 3+=13). The E75 V group had 77 patients' pathologythat underwent IHC (0=7, 1+=25, 2+=26, 3+=19). A comparable percentageof C and V patients were in each IHC group (0 C=8.8% vs. V=9.1%; 1+C=26.3% vs. V=32.5%, 2+ C=42.1% vs. V=33.8%, 3+ C=22.8% vs. V=24.7%).

Demographics, prognostic factors, and treatment profiles per THC statusare presented in Table 5. There were two significant differences inprognostic factors for IHC status groups. IHC 1+ patients had a largerpercentage of T2-T4 tumors in the C group compared to the V group (66.7%vs. 30.8%, p=0.05). IHC 3+ C patients were all NP and 42.1% of Vpatients were NP (p=0.003).

TABLE 5 Demographics, prognostic factors, and treatment profiles ofpatients enrolled in E75 Phase II trial by HER2/neu expression level. 00 1* 1* 2* 2* 3* 3* Control Vaccine Control Vaccine Control VaccineControl Vaccine (n = 5) (n = 7) p (n = 15) (n = 25) p (n = 24) (n = 26)p (n = 13) (n = 19) p Median age, 50 60 54 54 50 57 49 51 years Rangeyears 38-74 31-74 0.4 44-82 42-71 0.4  31-75 27-77 0.2 31-74 37-62 0.2Race White, % 100.0% 71.4% 0.5 73.3% 84.0% 0.4  87.5% 92.3% 0.7  61.5%89.5% 0.1 Other, %  0.0% 28.6% NS 26.7% 16.0% NS 12.5%  7.7% NS  40.5%10.5% NS Tumor size T2-T4, %  40.0% 14.3% 0.5 66.7% 28.0% 0.05 29.2%46.2% 0.2  38.5% 36.8% 0.8 Histological grade Grade III, %  20.0% 14.3%0.6 33.3% 36.0% 0.7  37.5% 38.5% 0.9  61.5% 57.9% 0.8 Node Positive 0.0%  0.0% NS 80.0% 60.0% 0.3  79.2% 80.8% 0.8 100.0% 42.1%   0.003*(NP), % Hormone  20.0% 14.3% 0.6 13.3% 28.0% 0.4  16.7% 11.5% 0.9  38.5%63.2% 0.2 receptor-, % Chemotherapy,  80.0% 42.9% 0.3 80.0% 76.0% 0.9 87.5% 96.2% 0.5  92.3% 94.7% 0.6 % XRT, % 100.0% 42.9%  0.08 66.7% 76.0%0.7  87.5% 96.2% 0.5  69.2% 94.7% 0.1 Hormonal  80.0% 85.7% 0.6 80.0%72.0% 0.9  79.2% 73.1% 0.6  53.8% 73.7% 0.3 therapy, % Herceptin, % 0.0%  0.0% NS  0.0%  0.0% NS  8.3%  7.7% 0.9  7.7% 10.5% 0.7*Statistically significant difference.

Immunologic Response per IHC Status:

The E75 vaccine was capable of eliciting an in vitro immune response inall IHC categories. All IHC groups (0, 1+, 2+, 3+) responded to thevaccine as noted by significant increases from pre-vaccine to maxE75-specific CD8+ T cells (0 p=0.007, 1+ p<0.001, 2+ p=0.004, 3+p=0.002). Only IHC 1+ patients trended towards significant pre tolong-term increase in E75-specific CD8+ T cells (p=0.08) (FIG. 12A).

In addition all patients were able to elicit an in vivo immune responseas measured via DTH pre- and post-vaccine. Significant pre-post DTHincreases were noted in all IHC categories (0 p=0.03, 1+ p=0.02, 2+p=0.02, 3+ p=0.05). Overall, regardless of HER2/neu expression asmeasured by IHC, the vaccine was immunologically effective but appearsmost effective in the IHC 1+ patients (FIG. 12B).

Clinical Response per IHC Status:

Clinical response, evaluated by recurrence and mortality, is noted inFIGS. 12C and 12D. In all IHC categories (except IHC 0 where no patientsrecurred), there were decreased recurrence rates when comparing C and Vpatients, although the numbers do not achieve statistical significance.More importantly, there was a significant decrease in mortality in IHC1+ patients, C=20% and V=0% mortality (p=0.04).

In a previous phase II trial, administration of the E75 vaccine resultedin decreased recurrence rates and a trend towards decreased mortalityrates at 20 months, but these differences lost significance as immunitywaned without the use of boosters. It was shown that patients having alllevels of HER2/neu expression responded immunologically to the vaccine,but that the LE (and specifically IHC 1+) patients had more robustimmunologic responses, and derived the greatest clinical benefit withdecreased mortality. It was also shown that antigen naïve patientsrespond immunologically to the vaccine as well.

When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations and subcombinations of ranges specific aspects therein areintended to be included.

All publications and patent applications cited in this specification areherein incorporated by reference in their entirety for all purposes asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference for allpurposes.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications can be made thereto without departing from the spiritor scope of the appended claims.

1-34. (canceled)
 35. A method of inducing protective or therapeuticimmunity against recurrence of an epithelial malignancy in a subjecthaving a fluorescence in situ hybridization (FISH) rating of less thanabout 2.0 for HER2/neu gene expression, said method comprisingadministering to the subject an effective amount of a compositioncomprising a pharmaceutically effective carrier and a peptide having theamino acid sequence SEQ ID NO:2.
 36. A method of inducing protective ortherapeutic immunity against recurrence of a HER2/neu expressing tumorin a subject, wherein the tumor is characterized as having afluorescence in situ hybridization (FISH) rating of less than about 2.0for HER2/neu gene expression, said method comprising administering tothe subject an effective amount of a composition comprising apharmaceutically effective carrier and a peptide having the amino acidsequence SEQ ID NO:2.
 37. A method of inducing protective or therapeuticimmunity against breast cancer recurrence in a subject having an afluorescence in situ hybridization (FISH) rating of less than about 2.0for HER2/neu gene expression, said method comprising administering tothe subject an effective amount of a composition comprising apharmaceutically effective carrier and a peptide having the amino acidsequence SEQ ID NO:2.
 38. A method of inducing protective or therapeuticimmunity against recurrence of HER2/neu expressing tumor in a subject,wherein the tumor is characterized as a fluorescence in situhybridization (FISH) rating of less than about 2.0 for HER2/neu geneexpression, said method comprising administering to the subject aneffective amount of a composition comprising a pharmaceuticallyeffective carrier and a peptide having the amino acid sequence SEQ IDNO:2 or a variant thereof, wherein immunity is established by astatistically significant increase in the presence of CD8⁺ cellsspecific for the peptide in peripheral blood mononuclear cells of thesubject when compared to pre-vaccine levels.
 39. The method of claim 35,wherein the composition is administered by injection or inoculation. 40.The method of claim 39, wherein the injection is an intradermalinjection.
 41. The method of claim 40, wherein the composition isinjected in one or more split doses.
 42. The method of claim 41, whereinthe injection sites are located about 5 cm apart from each other. 43.The method of claim 35 wherein the composition is administered at leastthree to six times on a monthly basis until the protective immunity isestablished.
 44. The method of claim 35, further comprisingadministering to the subject a booster after the primary immunizationschedule is completed, the booster comprising an effective amount of avaccine booster composition comprising a pharmaceutically effectivecarrier and a peptide having SEQ ID NO:2, or variant thereof.
 45. Themethod of claim 44, wherein the booster composition is administered byinjection.
 46. The method of claim 45, wherein the injection is anintradermal injection.
 47. The method of claim 45, wherein the boostercomposition is injected in one or more separate doses.
 48. The method ofclaim 47, wherein the injection sites are located about 5 cm apart fromeach other.
 49. The method of claim 44, wherein the booster isadministered about every six or more months after the primaryimmunization schedule is completed.
 50. The method of claim 35, whereinthe subject is a human.
 51. The method of claim 50, wherein the humanexpresses human leukocyte antigen A2.
 52. The method of claim 50,wherein the human expresses human leukocyte antigen A3.
 53. The methodof claim 35, wherein the composition further comprises an adjuvant. 54.The method of claim 53, wherein the adjuvant is recombinant humangranulocyte macrophage-colony stimulating factor.
 55. The method ofclaim 44, wherein the vaccine booster composition further comprises anadjuvant.
 56. The method of claim 55, wherein the adjuvant isrecombinant human granulocyte macrophage-colony stimulating factor. 57.The method of claim 35, wherein the composition comprises 1 mg of thepeptide and between about 0.01 to 0.5 mg of human granulocytemacrophage-colony stimulating factor as an adjuvant.
 58. The method ofclaim 35, wherein the composition comprises 1 mg of the peptide andabout 0.25 mg of human granulocyte macrophage-colony stimulating factoras an adjuvant.